Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements

Abstract

BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5' flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5' flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3'-flanking region, since constructs containing 3'-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3'-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5'-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.